The present invention relates to a therapeutic medical device, as classified by the FDA, in the form of a solid foam, comprised of a dried hydrogel of hydrophilic-hygroscopic polymer, more particularly, freeze-dried hydrogel of a polymeric carbohydrate, such as acemannan, to be used as a wound/lesion dressing, a drug delivery system, a hemostatic agent or a biological response modifier.
I. WOUND MANAGEMENT PA0 II. AVAILABLE PRODUCTS PA0 III. PHARMACOLOGICAL PROPERTIES OF POLYSACCHARIDES PA0 IV. PROPERTIES OF ACEMANNAN
Wound healing is a complex series of biochemical and cellular events which result in the contracting, closing and healing of a wound, a traumatic insult to the integrity of a tissue. Wound management must protect the wound from additional trauma and/or environmental factors that would delay the healing process.
Wound management usually consists of a combined systemic and local approach, including the use of antibiotics and the application of a suitable dressing. The principal function of a wound dressing is to provide an optimum healing environment. For example, a wound must be isolated from the external environment before healing can begin. A wound dressing covers the wound mimicking the natural barrier function of the epithelium. To provide an optimum healing environment, a wound dressing should control bleeding, protect the wound from the external environment, prevent further contamination or infection and maintain a moist micro-environment next to the wound surface.
Contamination of a wound may result from contact with an infected object or the ingress of dirt, dust, or microorganism, either at the time of injury or later from the patient's own skin or gastrointestinal tract. For example, it has been found that, unless effective measures are taken to prevent infection, virtually all burns become colonized by bacteria within 12 to 24 hours. In general, infection impedes wound healing by further damaging tissue and promoting inflammation. Subsequent further wound repair is delayed by the progression of inflammation consisting of vascular leakage, the release and activation of lyric enzymes, free radical generation, oxygen consumption, and the sensitization of tissue nerve endings. Thus any measure that limits inflammation should promote wound healing provided that it does not compromise the tissue's ability to resist infection and essential macrophage function.
Up to and including the late 1950's, it was generally accepted that, in order to prevent bacterial infection, a wound should be kept as dry as possible. However, a variety of studies have questioned this philosophy and found that wounds that were kept moist actually healed more rapidly than those that were left exposed to the air or covered with traditional dried dressings. In a review of the properties of occlusive dressings, W. H. Eaglestein, "The Effect of Occlusive Dressings on Collagen Synthesis and Re-epithelialization in Superficial Wounds," An Environment for Healing: The Role of Occlusion, Ryan, T. J. (ed.) International Congress and Symposium Series No. 88, London, Royal Society of Medicine, pp. 31-38 (1985) concluded that occlusive dressings that keep wounds moist could increase the rate of epidermal resurfacing by some 40%.
As a result of our greater understanding of the wound healing process, many new wound management products have been developed. Each of these products has its benefits and its deficits. In the case of large and/or irregular wounds, the available solid coverings such as gels, plastic, and gelatinous sheets generally do not maintain the close contact required for healing, especially for a wound with an irregular surface. Liquid gels cover the wound surface but are difficult to position and keep in place. In addition, they tend to become less stable at body temperature and flow out of the wound.
A. Absorbent Dressings
Semipermeable and impermeable wound dressings preserve the moisture in a wound but do not actively absorb excess moisture from the wound. The accumulation of wound fluid to the point of flooding can have severe consequences, including maceration and bacterial overgrowth. Dressings that are used to absorb exudate are frequently manufactured from cotton or viscous fibers enclosed in a sleeve of gauze. Such dressings are highly absorbent, but exhibit a tendency to adhere to the surface of the wound as fluid production diminishes. Furthermore, absorbent wound dressings generally do not provide adequate protection for the wound from the outside environment.
B. Nonadherent Dressings
Nonadherent dressings are designed not to stick to the wound. Gauze is often impregnated with paraffin or petroleum jelly to provide a nonadherent dressing. However, the impregnate can wear off, necessitating a dressing change and traumatizing new tissue growth.
In addition to the impregnated gauze type, nonadherent dressings may consist of an absorbent pad faced by a preformed nonadherent film layer.
C. Hydrogel Dressings
Hydrogels are complex lattices in which the dispersion medium is trapped rather like water in a molecular sponge. Available hydrogels are typically insoluble polymers with hydrophilic sites, which interact with aqueous solutions, absorbing and retaining significant volumes of fluid.
Hydrogel dressings are non-adherent and have a higher water content. Hydrogels have been reported to increase epidermal healing. Hydrogels progressively decrease their viscosity as they absorb fluid. In liquefying, hydrogels conform to the shape of the wound and their removal is untraumatic. However, currently available hydrogels are not biodegradable and do not consistently enhance the complete healing process.
D. Absorbable Materials
Absorbable materials are degraded in vivo and do not require removal. Particularly useful internally as hemostats, these materials include collagen, gelatin, and oxidized cellulose.
Gelfoam.RTM., an absorbable gelatin sponge, has been available and used in various surgical procedures as a topical hemostatic agent since the mid 1940's. Gelfoam.RTM., a brand of absorbable gelatin sterile sponge manufactured by Upjohn, is a medical device intended for application to bleeding surfaces as a hemostatic. It is a water insoluble, off-white, non-elastic, porous, pliable product prepared from purified porcine skin collagen. It can absorb and hold within its interstices, many times its weight of blood and other fluids. When not used in excessive amounts, Gelfoam.RTM. is absorbed completely, with little tissue reaction. This absorption is dependent on several factors, including the amount used, degree of saturation with blood or other fluids, and the site of use. When placed on soft tissues, Gelfoam.RTM. is usually absorbed completely in four to six weeks, without inducing excessive scar tissue.
The Physician's Desk Reference (1993 edition) suggests that one use only the minimum amount of Gelfoam.RTM. sterile sponge needed to achieve hemostasis, holding it at the site of injury until bleeding stops. Once hemostasis is reached, one should carefully remove any excess Gelfoam.RTM. as it may interfere with the healing of skin edges. Furthermore, Gelfoam.RTM. must not be placed in intravascular compartments because of the risk of embolization.
In addition, Gelfoam.RTM. is not recommended for use in the presence of an infection. If signs of infection or abscess develop where Gelfoam.RTM. has been positioned, reoperation may be necessary in order to remove the infected material and allow drainage.
Another precaution is that Gelfoam.RTM. should not be used in conjunction with autologous blood salvage circuits as it has been demonstrated that fragments of microfibular collagen pass through the 40 micron transfusion filters of blood scavenging systems.
E. Polysaccharide Dressings
One of the oldest and most enduring materials used in wound management is honey, a complex mixture consisting principally of glucose and fructose. Honey has a low pH, about 3.7, which creates an unfavorable environment for bacterial growth. However, honey has a high osmotic pressure and will effectively draw water out of the surrounding tissue and may dehydrate regenerating epithelial cells.
In recent years, there has also been an increasing interest in the use of sugar, sucrose, as a wound dressing. However, commercial sugar supplies are not always sterile and may contain calcium phosphate, sodium aluminum silicate, or other salts. Although the topical use of sugar appears to be relatively free of adverse effects, sugar has not been shown to be effective as the sole treatment of wounds in controlled clinical tests and may tend to dehydrate epithelial cells, macrophages and fibroblasts.
Available polysaccharide dressings such as Debrisan.TM., a linear polymer of glucose manufactured by Pharmacia, come formed into beads or granules that are poured into a wound and covered with a simple dressing pad or a semipermeable plastic film. The mobile nature of the beads can make Debrisan.TM. difficult to use in a shallow wound although the beads do provide a highly absorbent material that is biodegradable.
There are many examples in the literature indicating that polysaccharides can exhibit pharmacological and physiological activities without help from other components. Gialdroni-Grassi, International Archives of Allergy and Applied Immunology, 76 (Suppl. 1):119-127 (1985); Ohno et al., Chemical and Pharmaceutical Bulletin, 33(6):2564-2568 (1985); Leibovici et al., Chemico-Biological Interactions, 60:191-200 (1986); Ukai et al., Chemical and Pharmaceutical Bulletin, 31:741-744 (1983); Leibovici et al., Anticancer Research, 5:553-558 (1985). One such example relates to development of atherosclerosis. Hyperlipidemia in the general population and especially in familial hypercholesterolemia is associated with coronary heart disease and death. In countries where dietary fiber intake is high, atherosclerosis appears to be uncommon. Trowell et al., Editors, Refined Carbohydrate Foods and Disease, London, Academic Press, 207 (1975). Pectin and guar are reported to lower cholesterol in normal and hyperlipidemic patients. Kay et al., American Journal of Clinical Nutrition, 30:171-175 (1977). Locust bean gum, a polysaccharide composed of mannose and galactose, decreased the plasma lipoprotein cholesterol concentrations in both normal and familial hypercholesterolemic subjects. Zavoral et al., American Journal of Clinical Nutrition, 38:285-294 (1983). Addition of guar gum to carbohydrate meals decreased the postprandial rise of glucose in both normal and diabetic subjects. Jenkins et al., Lancet, 2:779-780 (1977). Kuhl, et al., in Diabetes Care, 6(2):152-154 (1983) demonstrated that guar gum exhibited glycemic control of pregnant insulin-dependent diabetic patients.
The antitumor activity of polysaccharides has been widely reported. Polysaccharides prepared from Lentinus cyathiformis are known to increase host defense against tumors. Rethy et al., Annales Immunologia Hungaricae, 21:285-290 (1981). There are several reports that polysaccharides from mushroom, yeast or bacterial extracts can elicit a high degree of host defense activity against vital and tumor infestations. Chihara, Nature, 222:687 (1969); Shwartzman et al., Proceedings of the Society for Experimental Biology and Medicine, 29:737-741 (1932); Suzuki et al., Journal of Pharmacobio-Dynamics, 7(7):492-500 (1984), also reported antitumor activity of a polysaccharide fraction (GF-1) extracted from cultured fruiting bodies of a fungus. Grifola frondosa. This fraction showed equivalent, high levels of inhibiting activity when administered intraperitoneally (IP), intravenously (IV) or intratumorally (IT). However, oral administration (PO) was not effective. The GF-1 fraction also exhibited antitumor action against the solid form of Meth A fibrosarcoma and MM 46 carcinoma in mice. Lentinan, which is a 6-branched .beta.-1-3-1inked glucan similar to GF-1, was ineffective against Meth A fibrosarcoma. Chihara, "The antitumor polysaccharide Lentinan: an overview;" Manipulation of Host Defense Mechanisms; Ed. by Aoki et al, Excerpta Medica, North Holland, 1-16 (1981); Sasaki et al. , Carbohydrate Research, 47(1):99-104 (1976).
Synthesized branched polysaccharides were also reported to demonstrate anti-tumor activity. Matsuzaki et al., Makromol Chem., 186(3):449-456 (1985). Matsuzaki et al., [Makromol. Chem., 187(2):325-331 (1986)] synthesized branched polysaccharides, which showed significant activities, both .beta.-(1-4)-D-mannopyranose and .beta.-(1-4)-linked glucomannan. A partially acetylated linear .beta.-(1-3)-D-mannan extracted from fruit bodies of Dictyophoria indusiata Fisch, also exhibited antitumor activity. Hara, Carbohydrate Research, 143:111 (1982). It appears that antitumor action depends on the type of polymer main chain and its degree of polymerization, because .beta.-(1-3)-glucan-type polymers show higher antitumor activity than .beta.-(1-4)-glucan and hemicellulosic polymers. Matsuzaki et al., Makromol. Chem., 187:325-331 (1986). A carboxymethylated derivative of .beta.-(1-3)-glucan obtained from bacterial culture filtrate caused severe cell loss from established sarcoma 180 tumors within 2 hours after the injection of the derivative. Baba, Journal of Immunopharmacology, 8 (6):569-572 (1986). The same author observed a compensatory increase in polymorphonuclear leukocytes due to injection of the substance. Incidentally, bestatin, a dipeptide known to possess immune-modulating and antitumor activity [Ishizuka, Journal of Antibiotics, 32:642-652 (1980)], influenced neither the tumor yield nor the polymorphonuclear leukocyte count. Baba et al., supra.
There are numerous reports on the antitumor effect of sulfated polysaccharides, including heparin [Jolles et al., Ata Univ. Int. Cancer, 16:682-685 (1960); Suemasu et al., Gann, 61(2):125-130 (1970)], sulfated laminaran and dextran [Jolles et al., British Journal of Cancer, 17:109-115 (1963)]. Yamamoto et al., in Japanese Journal of Experimental Medicine, 54:143-151 (1984), reported enhancement of antitumor activity of a fucoidan fraction by further sulfation. The sulfated product demonstrated activity against L-1210 leukemia. The authors postulated that the mechanism of the antitumor action might be due partly to inhibition of invasive growth of L-1210 cells, as a result of electrostatic repulsion between the tumor cell and mesothelial cells. Yamamoto et al., supra. Polysaccharides with sulfate groups are also reported to be human T cell mitogens and murine polyclonal B cell activators. Sugawara et al., Microbiological Immunology, 28(7):831-839 (1984). Generally, homopolysaccharides of high molecular weight with sulfate groups possess these properties. Dorries, European Journal of Immunology, 4:230-233 (1974); Sugawara et al., Cell Immunology, 74:162-171 (1982).
It has been reported that glucan extracted from the yeast Saccharomyces cervisiae is a modulator of cellular and humoral immunity. Wooles et al., Science, 142:1079-1080 (1963). The extracted glucan also stimulated proliferation of murine pluripotent hematopoietic stem cells, granulocyte macrophage colony-forming cells and cells forming myeloid and erythroid colonies. Pospisil et al., Experientia, 38:1232-1234 (1982); Burgaleta, Cancer Research, 37:1739-1742 (1977). Maisin et al., [Radiation Research, 105:276-281 (1986)] also reported that IV administration of a polysaccharide induced protection of murine hematopoietic stem cells against x-ray exposure, thereby decreasing the mortality of the mice so exposed.
Lackovic et al., [Proceedings of the Society for Experimental Biology and Medicine, 134:874-879 (1970)], took yeast cell wall and extracted all constituent matter leaving only "mannans" that he found to be responsible for the induction of an interferon production by peritoneal leukocytes. The "purified mannans" alleged to be responsible for this physiologic response had a molecular weight of 5,500-20,000 Daltons. He theorized that mannans stimulated mouse peritoneal macrophages to produce .gamma.-interferon. He also stated that the mannans he isolated showed no toxicity and "they are poor antigens." There was no mention by Lackovic et al. of the use of these "purified mannans" for antiviral activity or for IL-1 stimulation. We submit that Lackovic et al.'s "purified mannans" comprised an assortment of unknown and unidentified substituted and unsubstituted mannans.
Seljelid et al., [Experimental Cell Research, 131(1):121-129 (1981)] have observed that insoluble or gel-forming glycans activated macrophages in vitro, whereas the corresponding soluble glycans did not. They postulated that the orientation in which the glycan was presented to the mononuclear phagocyte was decisive for activation. Bogwald, [Scandinavian Journal of Immunology, 20:355-360 (1984)] immobilized glycans that had a stimulatory effect on the macrophages in vitro. This led the authors to believe that the spatial arrangements of the glycan was decisive for the effect on the macrophages in vitro. A purified polysaccharide isolated from Candida albicans induced an antibody response by human peripheral blood lymphocytes in vitro. Wirz et al., Clinical Immunology and Immunmopathology, 33:199-209 (1984). Yet there were significant differences between the antiCandida antibodies in sera of normal and Candida-infected individuals. Wirz et al., supra.
As discussed above, the biological activities of polysaccharide materials recovered from plants, yeast and bacteria have demonstrated direct biological activities by eliciting an increase in host defense systems. This reaction is primarily manifested by increased host surveillance for other antigenic substances. Polysaccharides serve as adjuvants (DEAE, Dextran, etc.) and immunomodulators. They also can function as unique T-cell-independent antigens. Both cellular and humoral immunity may be affected, and increased phagocytosis of infectious organisms and tumor cells has been observed, as has enhanced production of immunoglobulins.
The structure of these immunologically active polysaccharides and the types of structural variations appear to be the factors that control their potency and toxicity. Their mode(s) of action remain poorly understood; however, recent evidence indicates that several polysaccharides induce lymphocytes and macrophages to produce a wide range of immunologically active substances. For example, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) appears to be the chemical portion of lipopolysaccharide (LPS) that provides the minimum signal for macrophage host defense activation [Lebbar et al., Eur. J. Immunol, 16(1):87-91 (1986)].
The antiviral activity of polysaccharides and polysaccharides linked to peptides has been observed. Suzuki et al., Journal of Antibiotics, 32:1336-1345 (1979). Suzuki et al, supra, reported an antiviral action of peptidomannan (KS-2) extracted from mycelial culture of Lentinus edodes. Both oral and intraperitoneal administration increased the peak serum interferon titer, which protected mice against vital infections. This was different from dextran phosphate (DP-40) [Suzuki et al., Proceedings of the Society for Experimental Biology and Medicine, 149(4):1069-1075 (1975)] and 9-methylstreptimidone (9-MS) [Saito et al., Antimier, Agent & Chemotherapy, 10(1):14-19 (1976)], which induced higher titers of interferon in mice only if administered IV or IP.
Other researchers have also reported effects of complex polysaccharides [Saeki et al., Japanese Journal of Pharmacology, 24(1):109-118 (1974)], glycoproteins [Arita et al., Journal of Biochemistry, 76(4):861-869 (1974)] and sulfated polysaccharides [Rocha et al., Biochemical Pharmacology, 18:1285-1295 (1969)].
Ukai et al., [Journal of Pharmacobio-Dynamics, 6(12):983-990 (1983)] noted activity of polysaccharides extracted from the fruiting bodies of several fungi. The polysaccharides demonstrated a significant inhibitory effect on carrageenan-induced edema in rats. One of the polymers, O-acetylated-D-mannan (T-2-HN), in addition, demonstrated a more marked inhibitory effect than phenylbutazone on scald hyperalgesia. Ukai et al., supra. The assertion that the polysaccharide is free from protein and lipids strongly suggests that the effect is due to the acetylated mannan only.
Mannans, including glucomannans and galactomannans, have long been used by man. For example, galactomannans, in the form of plant gums, are widely employed as binders for control of food texture. In addition, some mannans have exhibited significant therapeutic properties [Davis and Lewis, eds. Jeanes, A., Hodge, J., In: American Chemical Society Symposium, Series 15, Washington, D.C., American Chemical Society, 1975]. Practitioners of Japanese folk medicine have long believed that extracts of certain fungi have anticancer activity. On investigation, many of these extracts have been found to contain complex carbohydrates with immune-stimulating activity. These carbohydrates are usually polymers of mannose (mannans), glucose (glucans), xylose (hemicellulose), fructose (levans) and mixtures of these. Individual sugars may be bonded in different ways as chains may be branched or unbranched. Glucans have been the most widely studied of these immunostimulatory carbohydrates. It has become increasingly clear that even though they have no toxicity mannans are as effective, if not more effective, than glucans.
A. Purified from Aloe vera
Aloe is a member of the lily family, Harding. Aloes of the World: A Checklist, Index and Code, Excelsa 9:57-94 (1979). Aloe barbadensis Miller is generally recognized as the "true aloe" because of its wide use and, reportedly, most effective healing power. Although in Japan, Aloe arborescens Miller traditionally has been used as a folk remedy for various ailments ranging from gastrointestinal disorders to athlete's foot. Aloe vera is a perennial plant with turgid green leaves joined at the stem in a rosette pattern. The leaves of a mature plant may be more than 25 inches long with sawlike spikes along their margins.
Aloe vera contains two major liquid sources, a yellow latex (exudate) and the clear gel (mucilage). The dried exudate of Aloe barbadensis Miller leaves is referred to as aloe. The commercial name is Curacao aloe. It is composed mainly of aloin, aloe-emodin and phenols. Bruce, South African Medical Journal, 41:984 (1967); Morrow et al., Archives of Dermatology, 116:1064-1065 (1980); Mapp et al., Planta Medica, 18:361-365 (1970); Rauwald, Archives Pharmazie, 315:477-478 (1982). A number of phenolics, including anthroquinones and their glycosides, are known to be pharmaceutically active. Bruce, Excelsa, 5:57-68 (1975); Suga et al., Cosmetics and Toiletries, 98:105-108 (1983).
The mucilaginous jelly from the parenchymal cells of the plant is referred to as Aloe vera gel. There are generally no anthroquinones to decompose and cause discoloration of the gel unless the gel is contaminated by an improper processing technique. Aloe vera gel is about 98.5% water by weight. More than 60% of the total solid is made up of polysaccharides of carbohydrate origin. Organic acids and inorganic compounds, especially calcium oxalate, account for the remainder of the solid residue.
Whole leaves, exudates and fresh gels of Aloe plants have been used for a variety of human afflictions. Evidence of their use as a medicinal remedy can be traced to the Egyptians of 400 BC. Aloe vera was also used to embalm the dead, as well as to protect the embalmers from the death-causing agent. Other early civilizations used Aloe vera for skin care, to relieve insect stings and bites, to treat scratches and ulcerated skin, to promote wound healing, to prevent hair loss and as a purgative. Aloe vera was used in the traditional medicine of many cultures as an anthelmintic, cathartic and stomachic and was used inter alia for leprosy, burns and allergic conditions. Cole et al., Archives of Dermatology and Syphilology, 47:250 (1943); Chopra et al., Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research, New Delhi (1956); Ship, Journal of the American Medical Association, 238(16):1770-1772 (1977); Morton, Atlas of Medicinal Plants of Middle American Bahmas to Yucatan, Charles C. Thomas Publisher, 78-80 (1981); Diez-Martinez, La Zabila, Communicado NO. 46 Sobre Recursos Bioticos Potenciales del Pais, INIREB, Mexico (1981); Dastur, Medicinal Plants of India and Pakistan, D. B. Taraporevala Sons & Co., Private Ltd., Bombay 16-17 (1962).
Depending on the way the leaves are processed, mucilage and sugars are the major components of the dehydrated gel. The sugars found are galactose, glucose, mannose, rhamnose, xylose and uronic acids. Although reports conflict, the mucilage is mainly composed of mannan or glucomannan. Eberendu et al., The Chemical Characterization of Carrisyn.RTM. (in preparation); Mandal et al., Carbohydrate Research, 86:247-257 (1980b); Roboz et al., Journal of the American Chemical Society, 70:3248-3249 (1948); Gowda et al., Carbohydrate Research, 72:201-205 (1979); Segal et al., Lloydia, 31:423 (1968).
For a long time, the controversy over the identity of the active substance(s) in Aloe vera was not settled. It is therefore important to clearly distinguish between the components present in the gel and those found in the exudates. A majority of the gel is a mucilage of mainly polysaccharide nature with minor amounts of various other compounds. It has been observed that in some of the activities there may be some synergistic action between the polysaccharide base and other components. Leung, Excelsa, 8:65-68 (1978); Henry, Cosmetics and Toiletries, 94:42-43, 46, 48, 50 (1979). For example, several workers report that the effective components for wound healing may be tannic acid [Freytag, Pharmazie, 9:705 (1954)] and a kind of polysaccharide. Wound-healing compositions from Aloe arborescens extracts. Kameyama, Japanese Patent #7856995, (1979). Mackee, supra, noted that the gel, not the rind or the exudate, was responsible for the beneficial effects in the treatment of radiation burns, and he stressed the importance of using fresh leaves for effective treatment. Polysaccharides degrade with time, and certain molecular weight sizes may be necessary to elicit a specified pharmacological response. Goto, et al., Gann, 63:371-374 (1972).
Literature which reports that polysaccharides possess pharmacological and physiological activities continues to flood the pages of well-respected scientific journals. It is therefore logical that the mucilaginous gel of the Aloe vera plant, which is essentially a polysaccharide, holds the secret to Aloe vera's medicinal properties. The controversy over whether the polysaccharide is a glucomannan, mannan, pectin, or of some other composition, is resolved by a series of chemical purification steps. Yagi et al., [Planta Medica, 31(1):17-20 (1977)], using a slightly modified extraction method, isolated acetylated mannan (aloe mannan) from Aloe arborescens Miller var natalensis. Ovodova [Khim, Prior. Soedin, 11(1):325-331 (1975)], however, earlier isolated pectin as the main component of the same aloe species.
B. Chemical Properties of Acemannan
Carrisyn.RTM. is the brand name given by the assignee of the instant invention to the purified ethyl alcohol extract of the inner gel of the leaves of Aloe barbadensis Miller. The active component of Carrisyn.RTM. has been designated "acemannan" by the United States Adopted Name Council. Not less than 73% of Carrisyn.RTM. extract is acemannan: Carrisyn.RTM. extract comprises generally about 73% to 90% acemannan. Carrisyn.RTM. extract is generally produced by removing the outer sheath of the leaf, then removing and processing the inner filet or mucilage as follows: pH adjustment, ethanol extraction, freeze drying and grinding. See U.S. application Ser. No. 144,872 filed January 1988 (now U.S. Pat. No. 4,851,224), a continuation-in-part of U.S. application Ser. No. 869,261 (now U.S. Pat. No. 4,735,935), the disclosures of all of which are incorporated herein by reference. Processing in this manner predicts that essentially no covalent bonds are altered and therefore no toxic compounds or byproducts are created. These manufacturing steps were developed to overcome the inability of traditional aloe product producers to standardize and stabilize the polysaccharides.
Acemannan is a fluffy, white, amorphous slightly hygroscopic powder, which is poorly soluble in water and dimethyl sulfoxide and insoluble in most other organic solvents. This powder consists of linear .beta.(1-4)-D-mannosyl units. The polysaccharide is a long chain polymer interspersed randomly with acetyl groups linked to the polymer through an oxygen atom. The generic name for the polymer is acemannan. The degree of acetylation is approximately 0.91 acetyl groups per monomer as determined by the alkaline hydroxamate method. See Hestrin, Journal of Biological Chemistry, 180:240-261 (1949). Neutral sugars linkage analysis indicates that attached to the chain, probably through an .alpha.(1-6) linkage, is a D-galactopyranose in the ratio of approximately one for every 70 sugars. The 20:1 ratio of mannose to galactose indicates that galactose units are also linked together, primarily by a .beta.(1-4) glycosidic bond. The chemical structure of acemannan may be represented as follows: ##STR1##
C. Toxicology
The toxicological effects of acemannan have been studied in both in vivo and in vitro systems. Acemannan is not mutagenic or blastogenic in in vitro test systems. In vitro, the compound showed no detectable toxicity for H-9, MT-2 and CEM-SS lymphoid cells. In vivo toxicology studies on acemannan include a 91-day subchronic oral toxicity study in dogs, a 180-day chronic oral toxicity study in rats and a 180-day clinical trials in humans. In these studies, no toxic effects were noted in dogs receiving up to 825 mg/kg of acemannan per day for 91 days. No clinical, gross pathologic or toxic effects were noted in rats receiving up to 38.475 ppm acemannan in their feed for 180 days. No adverse clinical or toxic effects were noted in human patients receiving 800 mg per day of acemannan for 180 days in clinical trials.
In pilot studies, administration of acemannan to dogs caused an absolute monocytosis in blood samples taken for complete white blood cell counts and morphology differential. Within 2 hours after oral administration of high doses of acemannan, large activated monocytes appeared in circulation. A similar effect has been observed in humans.
A study was performed using human peripheral blood monocyte cell cultures and .sup.14 C-labeled acemannan to track the incorporation or absorption of acemannan into a biological system. In this study, detectable amounts of .sup.14 C-labeled acemannan were absorbed or ingested by human peripheral monocyte/macrophage cells. Peak incorporation occurred at 48 hours. At a concentration of 5 mg/ml, the .sup.14 C-labeled acemannan was not cytotoxic to the monocyte/macrophage cells, and the weight/volume (w/v) digested cell mass was 760 times greater than the w/v of the digested acemannan solution. These results suggest that the macrophage is capable of maintaining intracellular concentration of acemannan at very high levels that are not cytotoxic.
A pyrogen assay was per,formed in rabbits in accordance with the pyrogen test protocol outlined in the U.S.P. XXI. Biological Test [151] using a 1 mg/ml injectable solution of acemannan. More frequent temperature measurements were taken than specified in the U.S.P. because of the unknown systemic effects of injected acemannan. Temperature changes in test animals did not exceed minimum changes allowed by the U.S.P. protocol; therefore, the solution met the U.S.P. requirements for absence of pyrogens. Injected acemannan elicited a maximum body temperature increase of 0.3.degree. C. in one rabbit. This temperature rise occurred 90 minutes after injection. Acemannan is an inducer of IL-1 secretion by macrophages and monocytes in vitro. Since IL-1 is a potent pyrogen, this might explain the minimal, delayed temperature rise in this rabbit.
Twenty-four human subjects enrolled in and completed the study of the safety and tolerance of orally-administered acemannan. Clinical laboratory results showed that shifts out of the normal range occurred in the following: CO.sub.2 in seven subjects, cholesterol in three subjects, triglycerides in two subjects, phosphorous in one, hemoglobin in four, basophils in two, monocytes in three, eosinophils in three, lymphocytes in four, neutrophils in two, and one each in red and white blood cells. Small numbers of red and white blood cells were also found in the urine. None of these shifts was clinically relevant.
Immune profile results showed group differences between Day 1 to Day 7 values for the following: CD-16, CD4 (T-4), CD-8-Leu7, CD-4-CD-25, CD-8-CD-16, Leu7 and TQ-1. Mitogen responses were in the low range.
Vital signs did not appear to exceed normal ranges. There were no group differences in urine output. One subject in Group IV had diarrhea and loose stools during the study. One subject in Group I had loose stools during days 2 to 4 of the study. A total of 5 subjects reported a total of eight adverse events. All the events occurred in subjects receiving 1600 or 3200 mg oral acemannan daily for 6 days.
D. Pharmacological Properties of Acemannan
Aloe vera has enjoyed a long history of lay acceptance as possessing "curative" or "healing" qualities. Over the last few years, numerous books and articles meeting scientific standards have been written on Aloe vera. Organizations such as the International Aloe Vera Science Council and recognized medical institutions, through publications and case histories of physicians, veterinarians and other scientists, have given credence to the "aloe phenomenon." Aloe vera has been featured extensively in the field of dermatology, especially for treating radiation-caused skin conditions. Mackee, X-rays and Radium in the Treatment of Diseases of the Skin, 3rd Ed., Lea and Febiger, Philadelphia, 319-320 (1938); Rovatti et al., Industrial Medicine and Surgery, 28:364-368 (1959); Zawahry et al., Quotations From Medical Journals and Aloe Research, Ed. Max B. Skousen, Aloe Vera Research Institute, Cypress, Calif., 18-23 (1977); Cera et al., Journal of the American Animal Hospital Association, 18:633-638 (1982). The body of scientific literature documenting medical applications in digestive problems, as a virucidal, bactericidal and fungicidal agent and in gynecological conditions is extensive and has been adequately reviewed by Grindley et al., [Journal of Ethnopharmacology, 16:117-151 (1986)].
A number of pharmacology studies have been conducted on Aloe vera gel in recent times. Results have included more rapid healing of radiation burns [Rowe, J. Am Pharm. Assoc., 29:348-350 (1940)] and accelerated healing of wounds [Lushbaugh et al., Cancer, 6:690-698 (1953)]. Thermal burns treated with Aloe vera gel heal much faster than untreated burns [Ashley et al., Plast. Reconstr. Surg., 20:383-396 (1957). Rovatto, supra. Rodriguez-Bigas et al., J. Plast. Reconstr. Surg., 81:386-389 (1988)]. The gel is useful in treating leg ulcers [El Zawahry et al., Int. J. Dermatol, 12:68-73 (1973)] and in hastening post surgical healing (Payne, Thesis submitted to Faculty of Baylor University, Waco, Tex., MS Degree). Experimental evidence suggests that extracts of Aloe vera have anti-infectious properties [Solar, Arch. Inst. Pasteur Madagascar, 47:9-39 (1979)] and enhance phagocytosis [Stepanova, Fiziol, Akt. Veshchestva, 9:94-97 (1977)].
Acemannan has also been shown to be a potent stimulator of the immune system. Acemannan induces the production of Interleukin 1 (Il-1) and prostaglandin E.sub.2 (PGE.sub.2) in human peripheral blood adherent cells in culture. Acemannan has been shown to be effective as an adjuvant and immunoenhancer and can be effectively used to treat cancer, vital disease, and infections. See U.S. Pat. No. 5,106,616 and U.S. Pat. No. 5,118,673 and references cited therein, the disclosures of which are incorporated herein by reference. All of these patents and this patent application are also assigned to Cartington Laboratories, Inc.
Despite the known therapeutic properties of many polysaccharides, despite the availability of various gels for treating a wound or lesion, and despite the obtainability of certain "water-insoluble" foam devices, there is a need for a relatively dry and flexible foam-like therapeutic device that can promote the healing of a wound or lesion, that can act as a drug delivery system, that can act as a biological response modifier, and that can transform into a relatively clear hydrogel upon absorption of a fluid, either a therapeutic liquid/suspension or body fluid.